The vital role of parathyroid hormone (PTH) in regulating serum calcium levels, and the involvement of PTH-related peptide (PTHrP) in embryonic development as well as its identification as the causative agent of severe hypercalcemia associated with certain malignancies, underscore the need for a thorough understanding of how these peptides bind to and activate their receptor. The primary goals of the experiments proposed here are 1) to identify critical residues in the ligand and to determine how they contribute to conformation, receptor binding and receptor activation, and 2) to identify and characterize key residues in the receptor with which the ligand interacts. It is expected that many residues in PTH contribute to the complex array of receptor-ligand interactions. We shall initially focus on those residues in PTH which our scanning mutagenesis studies show to be important. We have developed methods utilizing cells transfected with cloned PTH receptors and short synthetic PTH analogs to distinguish whether critical binding residues are involved in long-range intramolecular interactions or shorter-range interactions, e.g. with nearby residues on the receptor. Mutations which disrupt long-range interactions are to be used to isolate intragenic, second-site suppressor mutations that correct the binding defect of the primary site mutation. Such pairs of mutations will genetically identify two interacting sites in the ligand. In similar studies involving PTH/PTHrP hybrid peptides and a collaboration with Dr. M. Weiss, we shall correlate alterations in function with alterations in structure using the methods of 2D NMR. the collection of PTH analogs generated in aim I will be screened for receptor-specific effects using structurally distinct receptors generated by site-directed mutagenesis or derived from the cloning experiments described in Dr. Juppner's proposal (Subproject I). We shall map the sites in the receptor which determine the observed specificity using chimeric receptors and subsequent point mutation analysis as we have done for [Arg2]-PTH. Key receptor sites will be evaluated by saturation mutagenesis and the resulting mutants will be screened for ligand-specific effects and for activation-constitutive and activation-defective phenotypes. the large ligand-binding region of the receptor, which has been roughly mapped to between residues 1 and 300, will be systematically dissected using a series of deletions, followed by localized random mutagenesis and finally point mutation analysis. to complement these genetic approaches, we shall obtain direct evidence of specific ligand-receptor interactions by physically cross-linking a series of photoderivatized ligands to the receptor. The cross-linked sites will be characterized by fragmenting the complex with CNBr and sequencing the SDS-gel-purified label fragment. These studies should provide a substantial amount of new information about how PTH and PTHrP bind to and activate their common receptor. The data should thus lay the groundwork for the rational design of novel analogs which can be used to pharmacologically control the complex biologic effects of these potent peptides.